Filbertone-Induced Nrf2 Activation Ameliorates Neuronal Damage via Increasing BDNF Expression

Abstract

Neurotrophic factors are endogenous proteins that promote the survival of various neuronal cells. Increasing evidence has suggested a key role for brain-derived neurotrophic factor (BDNF) in the dopaminergic neurotoxicity associated with Parkinson's Disease (PD). This study explores the therapeutic potential of filbertone, a bioactive compound found in hazelnuts, in neurodegeneration, focusing on its effects on neurotrophic factors and the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway. In our study, filbertone markedly elevated the expression of neurotrophic factors, including BDNF, Glial cell line-Derived Neurotrophic Factor (GDNF), and Nerve Growth Factor (NGF), in human neuroblastoma SH-SY5Y cells, mouse astrocyte C8-D1A cells, and mouse hypothalamus mHypoE-N1 cells. Moreover, filbertone effectively countered neuroinflammation and reversed the decline in neurotrophic factors and Nrf2 activation induced by a high-fat diet (HFD) in neurodegeneration models. The neuroprotective effects of filbertone were further validated in models of neurotoxicity induced by palmitic acid (PA) and the neurotoxin MPTP/MPP⁺, where it was observed to counteract PA and MPTP/MPP⁺-induced decreases in cell viability and neuroinflammation, primarily through the activation of Nrf2 and the subsequent upregulation of BDNF and heme oxygenase-1 expression. Nrf2 deficiency negated the neuroprotective effects of filbertone in MPTP-treated mice. Consequently, our finding suggests that filbertone is a novel therapeutic agent for neurodegenerative diseases, enhancing neuronal resilience through the Nrf2 signaling pathway and upregulation of neurotrophic factors.

Keywords: Filbertone; Heme oxygenase-1; Neuroinflammation; Neurotrophic factors; Nuclear factor erythroid 2-related factor 2; Parkinson’s disease.

Fig. 1

Filbertone enhances neurotrophic factors in human neuroblastoma SH-SY5Y cells and in mouse astrocyte C8-D1A cells and mHypoE-N1 cells. (A and B) SH-SY5Y cells (A), C8-D1A cells (B) were treated with filbertone (0, 5, 10, 20, 40, 80, 160 and 320 µM) for 24 h. Cell viability was determined by WST-8 assay. (C-F) SH-SY5Y cells (C) were incubated with filbertone (0, 2, 10, and 50 µM) for 24 h. mRNA levels of BDNF were measured by qRT-PCR. (D-F) C8-D1A cells were incubated with filbertone (0, 2, 10, and 50 µM) for 24 h. mRNA levels of BDNF, GDNF, and NGF were measured by qRT-PCR. Data are mean ± SD (n = 3); *p < 0.05, **p < 0.01, and ***p < 0.001, calculated by one-way ANOVA followed by Tukey’s test

Fig. 2

Filbertone counteracts the decrease in neurotrophic factors and the activation of Nrf2 caused by HFD in the brain (A-G) Six-week-old male C57BL/6 were fed a normal chow diet (NCD) or high fat diet (HFD) for 16 weeks with filbertone (Fil). (A) p-Nrf2, Nrf2, HO-1, and BDNF in midbrain were measured by western blotting. Quantification of protein expression is shown in the right panel. (B-D) The expression of BDNF (B), NGF (C), GDNF (D) mRNA in midbrain were determined by qRT-PCR. (E-G) In hypothalamus, the expression levels of neurotrophic factors, BDNF (E), NGF (F), and GDNF (G) were detected by qRT-PCR. Data represent mean ± SD; *p < 0.05, **p < 0.01 and ***p < 0.001, calculated by one-way ANOVA followed by Tukey’s test

Fig. 3

Filbertone attenuates neuronal inflammation and the activation of glial cells in the brain (A and B) In midbrain, expression levels of inflammatory cytokines, TNF-a (A) and IL-1b (B) were detected by qRT-PCR. (C-E) In hypothalamus, the expression levels of inflammatory cytokines, TNF-a (C), IL-1b (D), and MCP-1 (E) were detected by qRT-PCR. (F-H) In hypothalamus, the expression levels of microglia activation markers, CD68 (F), CD11b (G), and Iba-1 (H) were detected by qRT-PCR. (I and J) The expression levels of astrocyte activation marker GFAP (I) and neuron damage marker HSP72 (J) were detected by qRT-PCR. Data represent mean ± SD; *p < 0.05, **p < 0.01 and ***p < 0.001, calculated by one-way ANOVA followed by Tukey’s test

Fig. 4

Filbertone-mediated Nrf2 activation protects against PA-induced neurotoxicity through the increase of BDNF and HO-1. To determine effects of filbertone (Fil) on palmitic acid (PA)-induced neurotoxicity in SH-SY5Y cells, the cells were incubated with filbertone (20 µM, 1 h) followed by PA (250 µM, 24 h). (A-D) Expression levels of BDNF (A), HO-1 (B), TNF-a (C), and IL-6 (D) were measured by qRT-PCR. (E) Viability of SH-SY5Y cells, the cells treated with filbertone and PA. The viability was detected by WST-8 assay. (F) Viability of SH-SY5Y cells. The cells were incubated with ZnPP (100 nM) in the presence of filbertone, followed by PA. (G) Viability of C8-D1A cells treated with filbertone and PA. (H) Viability of C8-D1A cells. The cells were incubated with ZnPP in the presence of filbertone, followed by PA. (I) Viability of mHypoE-N1cells, the cells treated with filbertone and PA. (J) Viability of mHypoE-N1 cells. The cells were incubated with ZnPP in the presence of filbertone, followed by PA. (K) Representative immunoblots for SH-SY5Y cells treated with filbertone and PA. Quantification of protein expression is shown in the right panel. Data represent mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, and NS; not significant, calculated by one-way ANOVA followed by Tukey’s test

Fig. 5

Filbertone-mediated activation of Nrf2 protects against neurotoxicity induced by MPTP/MPP⁺through the induction of BDNF and HO-1 (A-D) Six-week-old male C57BL/6 were intraperitoneally (i.p.) injected with MPTP (25 mg/kg) or vehicle. Five days before treatment with MPTP, mice received 0.2% filbertone treatment (A). (B) tyrosine hydroxylase (TH) and a-syn midbrain were measured by western blotting and densitometric analysis. (C-D) p-Nrf2, Nrf2, HO-1, and BDNF were measured by western blotting (C). Quantification of protein expression is shown in (D). (E) Viabilities of SH-SY5Y cells were detected by WST-8 assay. The cells were incubated with filbertone, followed by MPP⁺. (F) Viabilities of SH-SY5Y cells. The cells treated with MPP⁺ in the presence of filbertone and ZnPP (100 nM). (G) Representative immunoblots for SH-SY5Y cells treated with filbertone and MPP⁺. Quantification of protein expression is shown in the right panel. (H) Viabilities of C8-D1A cells were detected by WST-8 assay. The cells were incubated with filbertone, followed by MPP⁺. (I) Viabilities of C8-D1A cells. The cells treated with MPP⁺ in the presence of filbertone and ZnPP (100 nM). (J) Viabilities of mHypoE-N1 cells were detected by WST-8 assay. The cells were incubated with filbertone, followed by MPP⁺. (K) Viabilities of mHypoE-N1 cells. The cells treated with MPP⁺ in the presence of filbertone and ZnPP (100 nM). (L-O) The cells incubated with filbertone (20 µM, 1 h), followed by MPP+ (500 µM, 24 h) in mHypoE-N1 cells. Expression levels of BDNF (L), HO-1 (M), TNF-a (N), and IL-6 (O) were measured by qRT-PCR. Data represent mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, calculated by one-way ANOVA followed by Tukey’s test

Fig. 6

The deficiency of Nrf2 negates the protective effects of filbertone against neurotoxicity in SH-SY5Y and MPTP-treated mice (A) SH-SY5Y cells were incubated with filbertone (0, 5, 10, 20, and 40 µM) for 24 h. p-Nrf2, Nrf2, and HO-1 were measured by western blotting. Quantification of protein expression is shown in the right panel. (B) C8-D1A cells were incubated with filbertone (0, 5, 10, 20, and 40 µM) for 24 h. The mRNA levels of HO-1 were detected by RT-PCR. (C) mHypoE-N1 cells were incubated with filbertone (0, 5, 10, 20, and 40 µM) for 24 h. The mRNA levels of HO-1 were detected by RT-PCR. (D) Protein expression of BDNF in SH-SY5Y cells. BDNF were measured by western blotting. Quantification of protein expression is shown in the right panel. (E) The protein expression of BDNF, HO-1, p-Nrf2, and Nrf2 in SH-SY5Y cells were detected by western blotting. Quantification of protein expression is shown in the right panel. (F) The mRNA levels of BDNF, HO-1, and Nrf2 in SH-SY5Y cells were measured by RT-PCR. (G) BDNF and tyrosine hydroxylase (TH) expression in the mouse midbrain were detected by western blotting. Quantification of protein expression is shown in the right panel. Data represent mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, NS; not significant, calculated by one-way (A-D) and two- way (E-G) ANOVA followed by Tukey’s test